P1419: APEX1 NUCLEASE AND REDOX FUNCTIONS ARE BOTH ESSENTIAL FOR ADULT HEMATOPOIETIC STEM AND PROGENITOR CELLS.
Authors: S. Zaunz, L. Lauwereins, J. De Smedt, L. Cleuren, M. Bajaj, J. Marteijn, K. De Keersmaecker, C. Verfaillie
Journal: HemaSphere (2022)

Background: Hematopoietic stem (and progenitor) cells (HS(P)Cs) are highly vulnerable to enhanced reactive oxygen species levels, potentially leading to early hematopoietic senescence and/or malignant transformation. Apurinic/apyrimidinic endonuclease/redox factor 1 (APEX1) is a multifunctional protein that is essential for Base Excision Repair (BER), but also for gene transcription regulation, mainly involving the APEX1 redox signaling activity (Ref-1). Although BER is involved in repairing oxidized bases, as well as other spontaneous and frequent single base lesions, the role(s) of the core BER genes, such as Apex1, in maintaining adult HSPC functionality remain(s) to be elucidated. Similarly, its Ref-1 function, which reduces oxidized transcription factors and thereby regulates transcriptional networks, has not been studied in primary adult hematopoietic cells. Aims: We aim to study the role of the APEX1, often dysregulated in cancer, and its 2 functionally distinct domains in murine adult HSPCs. Methods: We assessed the role of the Apex1 gene in murine HSPCs using CRISPR-Cas9 knockout with 2 Apex1 targeting sgRNAs. The effect thereof was evaluated on hematopoietic repopulation in vivo as well as on HSPC expansion during long-term in vitro culture, using a HSPC maintaining medium (Wilkinson et al, 2020). Next, we delineated the role of the 2 functionally different domains of APEX1 in adult HSPCs, by culturing BM HSPC for 1 week in the continuous presence of different chemicals blocking the nuclease (involved in BER) or the Ref-1 function. To shed light on the general transcriptional changes caused by APEX1 deficiency, bulk RNA sequencing was performed on progeny of HSPC treated with or without inhibitors. Weighted gene co-expression analysis (WGCNA) was used to identify common and distinct gene clusters modified by different concentrations of the inhibitors. Results: We showed that full APEX1 KO HSPCs failed to repopulate irradiated recipient mice. Already after 2 weeks, significantly less APEX1 deficient cells were detected in the different blood lineages and nearly no KO cells could be detected from 4 weeks onwards. This was confirmed in vitro, where reduced expansion of APEX1 KO cells was observed. We demonstrated that both nuclease and Ref-1 APEX1 inhibitors lead to an ex vivo expansion defect of HSPCs, enhanced apoptosis and decreased cell proliferation rates. Bulk RNAseq analysis demonstrated that following nuclease and Ref-1 chemical inhibitions, immune response and interferon signaling were downregulated, whereas oxidative stress response genes were upregulated. Upon nuclease inhibition, genes involved in RNA and DNA metabolism were dysregulated, and integrin and thrombin signaling genes were upregulated. Although no specific module could be linked uniquely to Ref-1 inhibition, the majority of differentially expressed genes were involved in immune response and interferon signaling in contrast to the nuclease inhibition. Currently, we have completed single cell RNA sequencing which should identify the transcriptional networks regulated by the different APEX1 functions in the individual HSPC sub-populations. Summary/Conclusion: APEX1 and both its nuclease and its Ref-1 functions are essential for functional HSPCs. Moreover, our studies are defining the transcriptional networks regulated by the APEX1 nuclease and Ref-1 functions in primary HSPCs.